Cardiovascular diseases such as atherosclerosis are compounded in individuals who are obese, and metabolic disease and cardiovascular diseases are tightly linked. In part due to the obesity epidemic, cardiovascular disease remains the highest cause of mortality in our country. The goal of this project is to understand how adipose tissue directly surrounding blood vessels, perivascular adipose tissue (PVAT), regulates the vascular microenvironment. During obesity, PVAT expands and becomes dysfunctional, and we propose that these changes establish an atherosusceptible environment that promotes vascular disease. In preliminary studies, we have analyzed both human and mouse PVAT and found elevated levels of Notch signaling. This is true in human PVAT derived from patients with advanced vascular disease undergoing coronary artery bypass grafting surgery, as well as in mice fed a high fat diet. We hypothesize that constitutive Notch signaling in PVAT in conditions of obesity or high fat feeding leads to changes in Sirt proteins, which in turn affect the secretion of paracrine factors from PVAT to the vessel wall. Our approaches to test this hypothesis include an in-depth proteomic study of human PVAT derived from individuals with different stages of vascular disease, and corresponding analysis of PVAT in mouse models of atherogenesis. The aims of this project are: Specific Aim 1. Identify unique protein signatures of human and mouse PVAT that define atheroresistant versus atherosusceptible microenvironments. We will define novel targets in the PVAT secretome that regulate vascular cells under conditions of high fat diet and atherogenesis. Identified targets that potentially are protective or could aggravate vascular dysfunction will be tested directly for effects on vascular cells in vitro. Specific Aim 2. Determine how elevated Notch signaling in PVAT during obesity affects its phenotype and vascular disease. We will use a mouse genetic approach to alter Notch signaling in PVAT, and combine that with an ApoE null mouse on a high fat diet to study the effect on PVAT biology and atherogenesis. PVAT phenotype will be assessed based on identity as brown, beige, or white fat-like; and by assessment of adipokine production and inflammation. Unique Notch targets may be identified by cross-referencing changes in protein signatures identified in aim 1. Finally, PVAT adipocyte progenitors and their differentiation capacity will be characterized under different high fat diet conditions leading to atherosclerosis. Specific Aim 3. Evaluate the hypothesis that Notch regulation of Sirt genes during obesity contributes to changes in PVAT phenotype and its paracrine signaling activity to the vessel wall. We will utilize in vivo analysis using human PVAT derived from coronary artery bypass graft surgery, mouse models of obesity, and ex vivo studies with isolated PVAT from mice differing in Notch activity and metabolic state.